Removal of Non-specific Reactions of LABScreen Single Antigen Class II Assay by Fetal Bovine Serum Treatment (2025)

Kiwook Jung*,
Minjeong Nam*,
Yun Ji Hong,
Eun Youn Roh,
Sue Shin,
Kyoung Un Park and
Eun Young Song⇑

Departments of Laboratory Medicine, Seoul National University College of Medicine, Seoul, Korea

Address correspondence to Eun Young Song, MD; Department of Laboratory Medicine, Seoul National University College of Medicine, 101 Daehang-no Jongno-gu, Seoul 03080, Korea; phone. 82 2 2072 2548; fax. 82-2-747-0359; e mail: eysong1{at}snu.ac.kr

↵* These authors contributed equally to this work.

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Abstract

Recently, the removal of false reaction from beads 10 (DRB1^*04:04), 30 (DRB1^*16:01), and 31 (DRB1^*16:02) by fetal bovine serum (FBS) treatment in LABScreen Single Antigen Class II Assay was reported. We aimed to confirm the reaction in many cases. Fifty-nine sera showed positivity on at least two among beads 10, 30 and 31 from Nov 2017 to Oct 2018 in Seoul National University Hospital were included. FBS treatment was performed on 59 sera, and Single Antigen Class II Assay was repeated. Among 59 cases, the negative conversion rates of DR16 (57/59, 96.6%), DR4 (37/39, 94.9%), and accompanied other antibodies, e.g.) DP19 (39/45, 86.7%) were very high. The prior use of intravenous immunoglobulin in a non-specific binding group was significantly higher than the sex, age-matched control group (P=0.005).

Key words

Anti-HLA antibody
single antigen bead assay
fetal bovine serum
intravenous immunoglobulin

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Introduction

Renal transplantation is still the most important tool in the treatment of end stage renal disease, and donor specific antibodies are the most important factor of graft outcome. Solid-phase testing is widely used for HLA antibody detection. Mean fluorescence intensity (MFI) values of positive beads are sometimes used for treatment decision, though it was not approved for quantitative use by Food and Drug Administration (FDA) [1].

However, many interfering factors that affect MFI values of beads were reported. Interference by inherent substances in the serum such as IgM and complement are well known. Interference by exogenous substances such as intravenous Immnuglobulin (IVIG) and anti-thymocyte globulin (ATG) have also been reported. Another pitfall of single antigen assay is its reactivity to denatured antigens [2-4].

In 2017 April, One Lambda released LABScreen Single Antigen Class II assay lot 01012, and noted non-specific reactivity qualities of bead 10 (DRB1^*04:04), 30 (DRB1^*16:01), and 31 (DRB1^*16:02) on the worksheet. There was one report that all false reactions disappeared when they were treated with fetal bovine serum (FBS) [5]. Since we had realized that those reactions were removed by FBS in November 2017, we treated FBS on all suspected cases. In December 2018, the lot of LABScreen Single Antigen Class II assay beads were changed to lot 01013. False reactions to DRB1^*04:04 (bead 12), DRB1^*16:01 (bead 32), and DRB1^*16:02 (bead 33) are not shown anymore, but there is still a risk that those kinds of reactions can happen. We report the results of FBS treatment in false reactions within our laboratory to help build an understanding of the causes for false reactions, and alert scientists of the risk associated with such reactions.

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Materials and Methods

Study population

From November 2017 to October 2018, 59 sera showed positive reactions on at least two beads group among bead groups 10 (DRB1^*04:04), 30 (DRB1^*16:01) and 31(DRB1^*16:02) via a LABScreen Single Antigen Class II Assay (One Lambda, Canoga Park, CA, USA). Clinical characteristics included fluorescent antinuclear antibody (FANA), anti-streptococcal O (ASO), rheumatoid factor (RF), and history of prior drug use (IVIG, Rituximab, ATG, or Basiliximab within 3 month) and were analyzed using electronic medical records. Sex and age matched sera of fifty-nine patients, which showed no reactivity at beads 10, 30, and 31. They were randomly selected as a control group. This study was approved by the Institutional Review Board of Seoul National University Hospital (IRB No. 1811-158-989).

Single Antigen Bead Assay and Fetal bovine serum (FBS) treatment

Single antigen bead assay was performed using a LABScreen Single Antigen Class II Assay (One Lambda, USA) according to manufacturer’s recommendation. In brief, 5 uL of LABScreen beads was incubated with 20 uL of test serum in each well of a 96-well plate for 30 min, in the dark at 20-25°C with gentle shaking. After incubation, 150 uL of 1X wash buffer was added to each well. The plate was covered with a tray seal and vortexed. After centrifugation at 1,300 g for 5 min, wash buffer was removed by flicking. The same washing step was performed with 200 uL of 1X wash buffer twice. One hundred uL of 1X PE-conjugated anti-human IgG was added to each well. The plate was covered with a tray seal, and incubated in the dark for 30 min at 20-25°C with gentle shaking. After 5 min of centrifugation at 1,300 g, the supernatant was removed by flicking, with 200 uL of 1X wash buffer, two more runs of washing steps were performed. Eighty uL of 1X PBS was added to each well. The plate was covered with a tray seal and vortexed. Data acquisition and analysis were performed with LABScan 100TM Flow analyzer (Luminex Corp., Austin, TX, USA). The positive antibody assignment was performed by HLA fusion program version 4.2 (One Lambda, USA), and baseline MFI≥1000 was used for the positive cut-off value.

On 59 sera, which showed positive reaction on at least two beads group among beads group 10, 30 and 31, FBS treatment was performed as previously reported [6]. Three uL of FBS (Gibco, Waltham, USA) was added to 60 uL of 59 sera and mixed thoroughly. After 30 min of incubation, sera were centrifuged for 5 min at 7000 rpm. The supernatant was used to repeat this with LABScreen Single Antigen Class II Assay. Positively assigned antibodies were compared by the HLA fusion program, before and after FBS treatment.

Statistical analysis

The comparison of clinical characteristics between non-specific binding groups and the age, sex-matched control groups were performed using a Chi-square test or Fisher’s exact test. The level of significance was determined as P<0.05. All statistical analyses were performed using R software version 3.5.1 (The R Foundation for Statistical Computing, Vienna, Austria).

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Results

Among 59 cases, where at least two beads of 10, 30, and 31 were positive, DR16 antigen was assigned in all cases by HLA fusion software version 4.2 (One Lambda, USA). The strength of the DR16 reaction was weak (MFI 1000-2999) in 54 cases and moderate (MFI 3000-9999) in 5 cases. After FBS treatment, 57 cases were converted to negative reactions and 2 cases were still positive (Table 1). Among 59 cases, DR4 was assigned positively in 39 cases. The strength of the reaction was weak (MFI 1000-2999) in all cases. After FBS treatment, 37 cases (37/39, 94.9%) were converted to negative, and 2 cases were still positive. Other than DR4 and DR16, there were many other serotypes that accompanied HLA antibodies and were converted to negative after FBS treatment. Accompanied antibodies with incidence rates larger than 3 are shown in Table 1. DP19 was accompanied in 45 cases, and after FBS treatment, 39 cases were converted to negative (86.7%). DQA1^*05:03 (14 cases) and DR1 (10 cases) were converted to negative in 9 cases (9/14, 64.3%), and 7 cases (7/10, 70%), respectively (Table 1).

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Table 1

Negative conversion of false reactions after fetal bovine serum treatment.

Among 59 cases, 10 cases of negative converted antibodies were donor HLA specific antibodies (DSA). Seven cases were DR4, one case was DR16, another was DR4+DQ8, and the other case was DR16+DQ5.

Clinical characteristics of fifty nine cases and their sex and age matched controls were compared in Table 2. The positive rates of three serologic markers (FANA, RF, ASO) or underlying diseases were not statistically different between two groups. The frequencies of prior use for Rituximab, ATG and Basiliximab within 3 months were not statistically different between two groups. The frequency of the prior use of IVIG within 3 months was significantly higher in non-specific binding groups compared to control groups (33.3% vs 10.2%, P=0.005).

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Table 2

Comparison of clinical characteristics between the non-specific binding group and the control group.

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Discussion

From this study, we confirm that many cases which previously reported negative conversions of beads 10, 30, and 31 (DR4 and DR16) by FBS treatment [5]. In addition, we found that other accompanied HLA antibodies, especially DP19, frequently show negative conversion by FBS treatment.

Previous reports suggest that the cause of false reactions are related to a heterophile antibody, while FBS treatment provides target antigens that block the binding of heterophile antibodies to beads [5]. Heterophile antibodies are naturally occurring polyspecific antibodies to external antigens, and can nonspecifically bind to various antigens with low affinity. Therefore, they can be the causes of interference in many immunoassays [7].

Additionally, IVIG was suggested as another cause of nonspecific binding to beads 10, 30, and 31 in our study. IVIG is the human fraction of IgG, therefore it can include some heterophile antibodies. Other drugs are chimeric antibodies (Rituximab, Basiliximab), or animal-derived (ATG), and might not affect the nonspecific binding which was removed by FBS.

In our study, other than previously reported DR4, DR16, many HLA antibodies showed non-specific reactions. Especially DP19, which was accompanied in 45 cases and negatively converted in 86.7% after FBS treatment. In December 2018, a new lot of Single Antigen Class II antibodies (lot No. 01013) were released. With this, nonspecific DR4 and DR16 reactions were improved. However, nonspecific DP19 reaction was still observed in our laboratory (Data not shown). Although the DP antigen is less important in transplantation, caution must be exerted for non-specific bindings.

Among 59 cases, 10 cases of the negatively converted antibodies were DSA. A false report of DSA can cause critical errors in donor selection, or patient management in transplantation [8-10]. If non-specific reactions of DR4 and DR16 were ignored in laboratories despite being warned, review sessions for those cases should be conducted, especially when DR4 and DR16 were DSAs. The review process should repeat LABScreen Single Antigen Class II assay with new lot 01013.

In summary, many cases where the FBS treatment reduce non-specific reactions were con-firmed. They were mainly related to beads 10, 30, and 31 (DR4 and DR16) of LABScreen Single Antigen Class II assay lot 01012. Many accompanied antibodies, including DP19, also showed non-specific reactions. Causes of non-specific reaction were suggested to be heterophile antibodies because they are removed by FBS treatment. IVIG was also related to the non-specific reaction. In the future caution should remain for the non-specific reaction by heterophile antibodies, and FBS treatment should be performed when it is suspected.

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